Methods for the diagnosis and risk assessment of plasmalogen deficiency mediated diseases of aging

ABSTRACT

The present invention relates to methods for the diagnosis and risk assessment of plasmalogen deficiency mediated diseases of aging. The present invention describes the relationship between plasmalogen biosynthesis dysfunction and the biochemical and clinical manifestations of age related disorders. Specifically the present invention describes an increased prevalence of colon cancer, prostate cancer, lung cancer, breast cancer, ovary cancer, kidney cancer, cognitive impairment and dementia in subjects suffering from adult onset plasmalogen biosynthesis disorder (AO-PBD).

FIELD OF INVENTION

The present invention relates to methods for the diagnosis ofplasmalogen deficiency mediated diseases of aging. The present inventiondescribes the relationship between plasmalogen biosynthesis dysfunctionand the biochemical and clinical manifestations of age relateddisorders. Specifically the present invention describes an increasedprevalence of colon cancer, prostate cancer, lung cancer, breast cancer,ovarian cancer, kidney cancer, cognitive impairment and dementia insubjects with decreased levels of plasmalogens.

BACKGROUND OF THE INVENTION

Accordingly, the present application describes the discovery of a lateor adult onset form of peroxisomal dysfunction in humans. The diseasemanifests in subjects of all ages but the incidence increases withincreasing age after age 50 and peaks in 60-69 year olds and decreasesthereafter. Subjects suffering from age related plasmalogen deficiencieshave abnormally low levels of circulating plasmalogens in their serumand an increased prevalence of colon cancer, prostate cancer, lungcancer, breast cancer, ovary cancer, kidney cancer, cognitive impairmentand dementia relative to subjects without age related plasmalogendeficiencies.

The biosynthesis of plasmalogens has been recently reviewed in detail¹.The first two steps in the plasmalogen biosynthesis pathway are carriedout exclusively in peroxisomes (see FIGS. 1 and 22 for the fullpathway). The free hydroxyl group of dihydroxyacetone phosphate (DHAP)is first acetylated by DHAP acyltransferase (DHAP-AT). The ether bond(plasmanyl) is then created by replacing the sn-1 acyl group with afatty alcohol by alkyl-DHAP synthase. Loss of function of either ofthese two enzymes through point mutations, impaired peroxisomaltargeting or due to general peroxisomal dysfunction results in a severeplasmalogen deficiency. The remaining key synthetic processes occur inthe endoplasmic reticulum (ER) where the sn-2 position is acylated andphosphoethanolamine is added to the sn-3 position to create plasmanylglycerlyphosphoethanolamine (GPE). The final step involves aplasmanyl-specific enzyme that desaturates the 1-O-alkyl ether to formthe vinyl ether (plasmenyl) GPE species, commonly referred to as PlsEtnor plasmenylethanolamine, also commonly known as ethanolamineplasmalogens. All cells in the body are capable of synthesizing thesemolecules.

Peroxisomes were first discovered in the late 1960's by de Duve². Sincethat time over 50 different biochemical pathways have been described tobe performed by peroxisomes³. The nine primary biochemical systems arelisted in Table 1. The first peroxisomal disease of humans(cerebro-hepato-renal syndrome of Zellweger) was clinically described in1964 by Bowen et al⁴. In 1973, it was discovered that these patients haddisturbed mitochondrial function and no functional peroxisomes⁵.Currently, there are seventeen human diseases that are characterized asperoxisomal in origin (reviewed by Wanders⁶, Table 2). The peroxisomaldisorders are not uniform. Different disorders present vastly differentbiochemical abnormalities with some of these abnormalities overlapping.Accordingly, the diseases can be characterized as being either adisorder of peroxisomal biogenesis in which there is a general, overallperoxisomal deficit or a disorder of a particular peroxisomal protein orenzyme system (Table 3).

Of the peroxisomal disorders currently characterized, only RhizomelicChondrodysplasia Punctata (RCDP) can be said to be caused by plasmalogendeficiency alone. The RDCP disorders are further grouped into type I,II, or III. All three RDCP types exhibit decreased levels ofplasmalogens in the plasma and a decreased de novo synthesis capacity ofplasmalogens in the liver. Peroxisomal function in these subjects isbelieved to be otherwise normal, except for decreased α-oxidation ofphytanic acid in type I RCDP.

Subject suffering from RCDP exhibit severe mental retardation anddysplasias of the bone which result in stunted growth among otherabnormalities. Subjects with RCDP show numerous neurologicalabnormalities, the most striking of which is delayed myelination^(7, 8),which is believed to be a direct result of decreased plasmalogensynthesis. Most subjects with RCDP do not live more than two years frombirth.

Dysplasia is an abnormality in the appearance of cells indicative of anearly step towards transformation into a neoplasia. Dysplasia is apre-neoplastic state of a cell. This abnormal growth is restricted tothe originating system or location, for example, a dysplasia in theepithelial layer will not invade into the deeper tissue, or a dysplasiasolely in a red blood cell line (refractory anaemia) will stay withinthe bone marrow and cardiovascular systems. The best known form ofdysplasia is the precursor lesions to cervical cancer, called cervicalintraepithelial neoplasia (CIN). This lesion is usually caused by aninfection with the human papilloma virus (HPV). Dysplasia of the cervixis almost always unsuspected by the woman. It is usually discovered by ascreening test, the pap smear. The purpose of this test is to diagnosethe disease early, while it is still in the dysplasia phase and easy tocure. Dysplasia is the earliest form of pre-cancerous lesion in which acell begins to change away from its normal form to an abnormal, lessdifferentiated form. Carcinoma in situ, meaning ‘cancer in place’,represents a final transformation of a dysplasia cell to cancer, thoughthe cancer remains local and has not moved out of the original site.Dysplasia is not cancer.

Cancer is a state where the cells have lost their tissue identity andhave reverted back to a primitive cell form that grows rapidly andwithout regulation. Invasive carcinoma is the final step in thissequence. It is a cancer which has invaded beyond the original tissuelayer and is also able to spread to other parts of the body(metastasize), starting growth of the cancer there and destroying theaffected organs. It can be treated, but not always successfully.However, if left untreated it is almost always fatal.

In summary, a selective deficiency in plasmalogen biosynthesis is knownto result in the clinical manifestation of severe neurological andcellular growth abnormalities. Furthermore, the survival disadvantage ofdecreased plasmalogen biosynthesis is severe.

It is well known that many diverse human diseases such as cancer,dementia, or decreased cognitive functioning increase in incidence withage. From an epidemiological and statistical perspective, these diseasesoften look very similar. However, from a clinical perspective, each ofthe cancers, dementias, and decreased cognitive functioning are verydifferent. Currently, the largest risk factor for these disorders is thesubject's age. Furthermore, it is well established that most cancers,dementias, and decreased cognitive functioning have a long prodromalphase (5-15 years) in which the disease is present but at a sub-clinicalmanifestation. There are few, if any, practical methods to accuratelyand precisely identify subjects with a clearly elevated risk.Accordingly, there is a tremendous need to be able to accuratelyidentify subsets of the general population subjects with biochemicalabnormalities that are causally linked to the known biochemical etiologyof chronic age-related disorders such as cancers, dementias anddecreased cognitive functioning and then treat these subjects with safeand well tolerated therapeutics that can correct the biochemicalabnormality and reduce the risk of disease occurrence in thissub-population.

Impaired membrane cholesterol regulation, membrane dynamics, muscarinicreceptor signal transduction, and APP processing are implicated tovarious degrees and to various symptoms and pathologies observed indementia and decreased cognitive functioning. The association of thesebiochemical systems and dementia are well established.Acetylcholinesterase (ACE) inhibitors act by increasing the retentiontime of ACh in the synaptic cleft and therefore increase muscarinicreceptor transduction⁹. Statins act by reducing cholesterol levels¹⁰.Amyloid lowering drugs are currently in development and clinical trialsfor removing the accumulation or to reduce the production of amyloidplaques. Therefore, the direct findings presented above, stronglyimplicate plasmalogen biosynthesis impairment in the etiology ofdementia and cognitive impairment. In applicant's co-pending applicationPCT/CA2007/000313 metabolites selected from phosphatidylcholine-relatedcompounds, ethanolamine plasmalogens, endogenous fatty acids, essentialfatty acids, lipid oxidations byproducts, and metabolite derivatives ofthese metabolic classes were found to be at lower levels in samples frompatients suffering from dementia. In the present invention a decrease inplasmalogens has been found in patients suffering from other age relateddiseases.

One of the defining features regarding cancer cells is that, unlikenormal cells which rely almost entirely upon respiration for energy,cancer cells can utilize both respiration and glycolysis for energy. Incancer, much work is now focused on developing drugs that inhibit theglycolysis pathway. One of the defining features of aerobic glycolysisin cancer is an enhanced mitochondrial citrate export and the use ofcytosolic citrate to form acetyl-CoA. Therefore, the direct findingspresented above, that an impairment in plasmalogen biosynthesis resultin both increased membrane cholesterol levels and increased cytosolicacetyl-CoA utilization, strongly implicate plasmalogen biosynthesisimpairment in cancer etiology.

The present application describes a subset of adult humans (>age 40) whohave abnormally low levels of plasmalogens in their serum. Thisdeficiency has been determined to be due to decreased plasmalogensynthesis and not due to increased oxidative stress. Subjects with thisdisorder have an increased prevalence of cognitive impairment, dementiaand cancer. The early diagnosis of these diseases, or the assessment ofrisk in subjects before they get these diseases will result in atremendous improvement on the long-term quality of life of thesesubjects as well as have a tremendous long-term cost saving to existinghealth care systems.

SUMMARY OF THE INVENTION

The present invention relates to methods for the diagnosis ofplasmalogen deficiency mediated diseases of aging. The present inventiondescribes the relationship between plasmalogen biosynthesis dysfunctionand the biochemical and clinical manifestations of age relateddisorders. Specifically the present invention describes an increasedprevalence of colon cancer, prostate cancer, lung cancer, breast cancer,ovarian cancer, kidney cancer, cognitive impairment and dementia insubjects with decreased levels of plasmalogens.

The present invention discloses a novel method of diagnosing thepresence of age-related plasmalogen deficiency in one or more than onesubject by measuring the levels of one or more than one plasmenyl orplasmanyl ether lipid present in a serum sample taken from a subject ofunknown disease status and comparing these levels to “normal” orage-related plasmalogen deficiency reference levels and through thiscomparison arriving at either an age-related plasmalogen deficiencypositive or age-related plasmalogen deficiency negative diagnosis.

The present invention discloses a novel method of diagnosing thepresence of age-related plasmalogen deficiency in one or more than onesubject by comparing a mathematically determined plasmalogen score fromthe measurement of one or more than one plasmenyl or plasmanyl etherlipid present in a serum sample taken from one or more than one subjectof unknown disease status and comparing this score to “normal” orage-related plasmalogen deficiency reference levels and through thiscomparison arriving at either an age-related plasmalogen deficiencypositive or age-related plasmalogen deficiency negative diagnosis.

The present invention discloses a novel method of diagnosing thepresence of age-related plasmalogen deficiency in one or more than onesubject by comparing the ratio of one or more than one plasmenyl orplasmanyl ether lipid to one or more than one endogenous moleculesunaffected or minimally affected by age-related plasmalogen deficiencyfrom serum samples taken from one or more than one subject of unknowndisease status and comparing these ratios to “normal” or age-relatedplasmalogen deficiency reference levels and through this comparisonarriving at either an age-related plasmalogen deficiency positive orage-related plasmalogen deficiency negative diagnosis.

Since subjects with an age-related plasmalogen deficiency have elevatedrisk of getting cancer and dementia, the present invention discloses anovel method for identifying subjects that are at elevated risk ofdeveloping cancer or dementia. Accordingly, the present inventiondiscloses a novel method of diagnosing an elevated risk of gettingcancer or dementia in one or more than one subject by measuring thelevels of one or more than one plasmenyl or plasmanyl ether lipidpresent in a serum sample taken from a subject of unknown disease statusand comparing these levels to “normal” or age-related plasmalogendeficiency reference levels and through this comparison arriving at adetermination of elevated risk or not.

Since subjects with an age-related plasmalogen deficiency have elevatedrisk of getting cancer and dementia, the present invention discloses anovel method for identifying subjects that are at elevated risk ofdeveloping cancer or dementia. Accordingly, the present inventiondiscloses a novel method of diagnosing an elevated risk of gettingcancer or dementia in one or more than one subject by comparing amathematically determined plasmalogen score from the measurement of oneor more than one plasmenyl or plasmanyl ether lipid present in a serumsample taken from one or more than one subject of unknown disease statusand comparing this score to “normal” or age-related plasmalogendeficiency reference levels and through this comparison arriving at adetermination of elevated risk or not.

Since subjects with an age-related plasmalogen deficiency have elevatedrisk of getting cancer and dementia, the present invention discloses anovel method of diagnosing an elevated risk of getting cancer ordementia in one or more than one subject by comparing the ratio of oneor more than one plasmenyl or plasmanyl ether lipid to one or more thanone endogenous molecules unaffected or minimally affected by age-relatedplasmalogen deficiency from serum samples taken from one or more thanone subject of unknown disease status and comparing these ratios to“normal” or age-related plasmalogen deficiency reference levels andthrough this comparison arriving at a determination of elevated risk ornot.

Since there is an increased prevalence of an age-related plasmalogendeficiency in subjects currently suffering from cancer or dementia, thepresent invention discloses a novel method for identifying subjects withundiagnosed cancer or dementia. Accordingly, the present inventiondiscloses a novel method of identifying undiagnosed cancer or dementiain one or more than one subject by measuring the levels of one or morethan one plasmenyl or plasmanyl ether lipid present in a serum sampletaken from a subject of unknown disease status and comparing theselevels to “normal” or age-related plasmalogen deficiency referencelevels. Subjects that test positive for an age-related plasmalogendeficiency are then tested by conventional cancer and dementiadiagnostic methods to determine the location of the cancer and/or theseverity and type of dementia present in said subject.

Since there is an increased prevalence of an age-related plasmalogendeficiency in subjects currently suffering from cancer or dementia, thepresent invention discloses a novel method for identifying subjects withundiagnosed cancer or dementia. Accordingly, the present inventiondiscloses a novel method of identifying undiagnosed cancer or dementiain one or more than one subject by comparing a mathematically determinedplasmalogen score from the measurement of one or more than one plasmenylor plasmanyl ether lipid present in a serum sample taken from one ormore than one subject of unknown disease status and comparing this scoreto “normal” or age-related plasmalogen deficiency reference levels.Subjects that test positive for an age-related plasmalogen deficiencyare then tested by conventional cancer and dementia diagnostic methodsto determine the location of the cancer and/or the severity and type ofdementia present in said subject.

Since there is an increased prevalence of an age-related plasmalogendeficiency in subjects currently suffering from cancer or dementia, thepresent invention discloses a novel method for identifying subjects withundiagnosed cancer or dementia. Accordingly, the present inventiondiscloses a novel method of identifying undiagnosed cancer or dementiain one or more than one subject by comparing the ratio of one or morethan one plasmenyl or plasmanyl ether lipid to one or more than oneendogenous molecules unaffected or minimally affected by age-relatedplasmalogen deficiency from serum samples taken from one or more thanone subject of unknown disease status and comparing these ratios to“normal” or age-related plasmalogen deficiency reference levels.Subjects that test positive for age-related plasmalogen deficiency arethen tested by conventional cancer and dementia diagnostic methods todetermine the location of the cancer and/or the severity and type ofdementia present in said subject.

The present invention provides a method for identifying an individualwho would benefit from an age-related plasmalogen deficiency-targetedtherapy comprising: analyzing a blood sample from a test subject toobtain quantifying data on all or a subset of the metabolites listed inTable 5, or closely related entities; comparing the data obtained onsaid metabolites in said test subject with reference data obtained fromthe analysis of a plurality of age-related plasmalogen deficiency humansor from a plurality of non-age-related plasmalogen deficiency humans;and using said comparison to determine the probability that the testsubject would benefit from an age-related plasmalogendeficiency-targeted therapy.

The present invention provides a method for monitoring the effect of anage-related plasmalogen deficiency-targeted therapy comprising:analyzing a plurality of blood samples from a test subject prior to theinitiation, during administration, or following administration of suchtherapy to obtain quantifying data on all or a subset of the metaboliteslisted in Table 5, or closely related entities; comparing the dataobtained on said metabolites in said samples to each other or toreference data obtained from the analysis of a plurality of age-relatedplasmalogen deficiency humans or from a plurality of non-age-relatedplasmalogen deficiency humans; and using said comparison to determinethe probability that the test subject would benefit from the continuedtreatment of an age-related plasmalogen deficiency-targeted therapy.

The impact of the present invention on the diagnosis of age-relatedplasmalogen deficiency and diseases caused by age-related plasmalogendeficiency would be tremendous, as literally everyone could be screenedlongitudinally throughout their lifetime to assess risk.

Given that the causal relationship between age-related plasmalogendeficiency and cancer and dementia is stronger than any previouslydescribed relationship and that the performance characteristics of themethods described in the present invention are representative for thegeneral population, these methods alone may be superior to any othercurrently available screening method for cancer, dementia and cognitiveimpairment, as it may have the potential to detect disease progressionprior to the emergence of clinical symptoms allowing for earlierintervention and subsequent better prognosis for subjects suffering fromthese diseases.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows the biosynthesis pathway for plasmalogens.

FIG. 2 shows the average ratio of plasmenyl 16:0/22:6 to diacyl16:0/18:0 across different diseases.

FIG. 3 shows the average ratio of plasmenyl 16:0/18:2 to diacyl16:0/18:0 across different diseases.

FIG. 4 shows the mean+/−SEM of diacyl 16:0/18:0 (M01), plasmanyl16:0/18:2 (M11), plasmenyl 16:0/18:2 (M16), plasmanyl 16:0/22:6 (M09),and plasmenyl 16:0/22:6 (M19).

FIG. 5 shows the distribution of plasmalogen concentrations inpopulation normals.

FIG. 6 shows the distribution of plasmalogen concentrations in cognitiveconfirmed normals.

FIG. 7 shows the distribution of plasmalogen concentrations in probableAD patients.

FIG. 8 shows the distribution of plasmalogen concentrations in renalcancer patients.

FIG. 9 shows the distribution of plasmalogen concentrations in prostatecancer patients.

FIG. 10 shows the distribution of plasmalogen concentrations in ovariancancer patients.

FIG. 11 shows the distribution of plasmalogen concentrations in lungcancer patients.

FIG. 12 shows the distribution of plasmalogen concentrations in coloncancer patients.

FIG. 13 shows the distribution of plasmalogen concentrations in breastcancer patients.

FIG. 14 shows the percentage of subjects with serum plasmalogen levelsless than 50% of normal levels.

FIG. 15 shows the effect of dementia severity and SDAT pathology onserum PlsEtn levels. (FIG. 15A) Mono and di-unsaturated PlsEtn. (FIG.15B) Polyunsaturated PlsEtn and free DHA (22:6). PlsEtn abbreviations:(fatty acid carbons: double bonds, not including the vinyl ether doublebond) and position on glycerol backbone (sn-1/sn-2). 22:6 representsfree DHA. Values are expressed as mean±SEM (n=19-112).

FIG. 16 shows the linear regression analysis of disease severity andserum PlsEtn 16:0/22:6 levels in 256 SDAT subjects. X=predicted time ofAO-PBD occurrence. Values are expressed as mean±SEM (n=66-112). Clinicalprogression assumes 7.5 ADAS-cog points/year.

FIG. 17 shows the etiology of dementia.

FIG. 18 shows the percentage of healthy subjects predicted to get cancerin the next 15 years.

FIG. 19 shows the percentage of healthy 50-59 and 60-69 year-olds withage-related plasmalogen dificiency, predicted to get dementia within 20years or cancer within 15 years, and the percentage of those casespredicted to be attributed to age-related plasmalogen dificiency.

FIG. 20 shows the distribution of plasmenyl 16:0/22:6 inautopsy-confirmed non-Alzheimer's subjects.

FIG. 21 shows the distribution of plasmenyl 16:0/22:6 inautopsy-confirmed Alzheimer's subjects.

FIG. 22 shows the biosynthetic pathway of plasmalogens in mammals.

FIG. 23 shows the Q-Trap flow injection analysis standard curve ofPlsEtn 16:0/22:6 in healthy human serum.

DETAILED DESCRIPTION

The present invention relates to methods for the diagnosis ofplasmalogen deficiency mediated diseases of aging. The present inventiondescribes the relationship between plasmalogen biosynthesis dysfunctionand the biochemical and clinical manifestations of age relateddisorders. Specifically the present invention describes an increasedprevalence of colon cancer, prostate cancer, lung cancer, breast cancer,ovarian cancer, kidney cancer, cognitive impairment and dementia insubjects with decreased levels of plasmalogens.

Accordingly, the present invention describes the discovery of a late oradult onset form of peroxisomal dysfunction in humans. The diseasemanifests in subjects of all ages but the incidence increases withincreasing age after age 50 and peaks in 60-69 year olds and decreasesthereafter. Subjects suffering from age related plasmalogen deficiencieshave abnormally low levels of circulating plasmalogens in their serumand an increased prevalence of colon cancer, prostate cancer, lungcancer, breast cancer, ovary cancer, kidney cancer, cognitive impairmentand dementia relative to subjects without age related plasmalogendeficiencies. The terms age-related plasmalogen deficiency or adultonset plasmalogen biosynthesis disorder, or AO-PBD have been usedthroughout this application to describe this disorder. Although theembodiments of this invention have been exemplified for increaseprevalence of colon cancer, prostate cancer, lung cancer, breast cancer,ovary cancer, kidney cancer, cognitive impairment or dementia, otherage-related plasmalogen deficiency disorders can be diagnosed, or therisk of acquiring said disorders can be assessed according to thepresent invention.

The diagnostic method of the present invention is minimally invasive andis indicative of AO-PBD. Translation of the method into a clinical assaycompatible with current clinical chemistry laboratory hardware iscommercially acceptable and effective. Furthermore, the method of thepresent invention does not require highly trained personnel to performand interpret the test.

The biological samples could originate from anywhere within the body,for example but not limited to, blood (serum/plasma), cerebral spinalfluid (CSF), urine, stool, breath, saliva, or biopsy of any solid tissueincluding tumor, adjacent normal, smooth and skeletal muscle, adiposetissue, liver, skin, hair, brain, kidney, pancreas, lung, colon,stomach, or other. Of particular interest are samples that are serum orCSF. While the term “serum” is used herein, those skilled in the artwill recognize that plasma or whole blood or a sub-fraction of wholeblood may be used.

When a blood sample is drawn from a patient there are several ways inwhich the sample can be processed. The range of processing can be aslittle as none (i.e. frozen whole blood) or as complex as the isolationof a particular cell type. The most common and routine proceduresinvolve the preparation of either serum or plasma from whole blood. Allblood sample processing methods, including spotting of blood samplesonto solid-phase supports, such as filter paper or other immobilematerials, are also contemplated by the invention.

The processed blood sample described above is then further processed tomake it compatible with the methodical analysis technique to be employedin the detection and measurement of the biochemicals contained withinthe processed serum sample. The types of processing can range from aslittle as no further processing to as complex as differential extractionand chemical derivatization. Extraction methods could includesonication, soxhlet extraction, microwave assisted extraction (MAE),supercritical fluid extraction (SFE), accelerated solvent extraction(ASE), pressurized liquid extraction (PLE), pressurized hot waterextraction (PHWE) and/or surfactant assisted extraction (PHWE) in commonsolvents such as methanol, ethanol, mixtures of alcohols and water, ororganic solvents such as ethyl acetate or hexane. The preferred methodof extracting metabolites for HTS analysis is to perform a liquid/liquidextraction whereby non-polar metabolites dissolve in an organic solventand polar metabolites dissolve in an aqueous solvent.

One embodiment of the present invention detects and measures a panel ofmetabolites in which a subset were found to have statisticallysignificantly differential abundances between AO-PBD and normal serum.In one embodiment the panel of metabolites is one or more than onemetabolites listed in Table 5.

The present invention provides a method for diagnosing AO-PBD or therisk of AO-PBD in a patient, the method comprising the steps of:

-   -   a) analyzing a sample from said patient to obtain quantifying        data for one or more than one metabolite marker;    -   b) comparing the quantifying data for said one or more than one        metabolite marker to corresponding data obtained from one or        more than one reference sample, wherein said comparison can be        used to diagnose AO-PBD or the risk of AO-PBD.

The step of analyzing the sample may comprise analyzing the sample usinga mass spectrometer (MS). For example, and without wishing to belimiting, such mass spectrometer could be of the FTMS, orbitrap, time offlight (TOF) or quadrupole types. Alternatively, the mass spectrometercould be equipped with an additional pre-detector mass filter. Forexample, and without wishing to be limiting such instruments arecommonly referred to as quadrupole-FTMS (Q-FTMS), quadrupole TOF (Q-TOF)or triple quadrupole (TQ or QQQ). In addition, the mass spectrometercould be operated in either the parent ion detection mode (MS) or in MSnmode, where n>=2. MSn refers to the situation where the parent ion isfragmented by collision induced dissociation (CID) or otherfragmentation procedures to create fragment ions, and then one or morethan one of said fragments are detected by the mass spectrometer. Suchfragments can then be further fragmented to create further fragments.Alternatively, the sample could be introduced into the mass spectrometerusing a liquid or gas chromatographic system or by direct injection.

The extracted samples may be analyzed using any suitable method know inthe art. For example, and without wishing to be limiting in any manner,extracts of biological samples are amenable to analysis on essentiallyany mass spectrometry platform, either by direct injection or followingchromatographic separation. Typical mass spectrometers are comprised ofa source which ionizes molecules within the sample, and a detector fordetecting the ionized molecules or fragments of molecules. Non-limitingexamples of common sources include electron impact, electrosprayionization (ESI), atmospheric pressure chemical ionization (APCI),atmospheric pressure photo ionization (APPI), matrix assisted laserdesorption ionization (MALDI), surface enhanced laser desorptionionization (SELDI), and derivations thereof. Common mass separation anddetection systems can include quadrupole, quadrupole ion trap, linearion trap, time-of-flight (TOF), magnetic sector, ion cyclotron (FTMS),Orbitrap, and derivations and combinations thereof. The advantage ofFTMS over other MS-based platforms is its high resolving capability thatallows for the separation of metabolites differing by only hundredths ofa Dalton, many which would be missed by lower resolution instruments.

By the term “metabolite”, it is meant specific small molecules, thelevels or intensities of which are measured in a sample, and that may beused as markers to diagnose a disease state. These small molecules mayalso be referred to herein as “metabolite marker”, “metabolitecomponent”, “biomarker”, or “biochemical marker”.

The metabolites are generally characterized by their accurate mass, asmeasured by mass spectrometry technique used in the above method. Theaccurate mass may also be referred to as “accurate neutral mass” or“neutral mass”. The accurate mass of a metabolite is given herein inDaltons (Da), or a mass substantially equivalent thereto. By“substantially equivalent thereto”, it is meant that a +/−5 ppmdifference in the accurate mass would indicate the same metabolite, aswould be recognized by a person of skill in the art. The accurate massis given as the mass of the neutral metabolite. As would be recognizedby a person of skill in the art, the ionization of the metabolites,which occurs during analysis of the sample, the metabolite will causeeither a loss or gain of one or more hydrogen atoms and a loss or gainof an electron. This changes the accurate mass to the “ionized mass”,which differs from the accurate mass by the mass of hydrogens (or otheradducts such as sodium, potassium, ammonia, and others known in the art)and electrons lost or gained during ionization. Unless otherwisespecified, the accurate neutral mass will be referred to herein.

Similarly, when a metabolite is described by its molecular formula themolecular formula of the neutral metabolite will be given. Naturally,the molecular formula of the ionized metabolite will differ from theneutral molecular formula by the number of hydrogens (or other adductssuch as sodium, potassium, ammonia, and others known in the art) lost orgained during ionization.

Data is collected during analysis and quantifying data for one or morethan one metabolite is obtained. “Quantifying data” is obtained bymeasuring the levels or intensities of specific metabolites present in asample.

The quantifying data is compared to corresponding data from one or morethan one reference sample. The “reference sample” is any suitablereference sample for the particular disease state. For example, andwithout wishing to be limiting in any manner, in the present inventionthe reference sample may be a sample from a non-AO-PBD controlindividual, i.e., a person not suffering from any age-relatedplasmalogen deficiency disease (also referred to herein as a “‘normal’counterpart”). As would be understood by a person of skill in the art,more than one reference sample may be used for comparison to thequantifying data.

In yet another embodiment of the present invention, there is provided amethod for diagnosing AO-PBD or the risk of AO-PBD in a patient. Themethod comprising the steps of:

-   -   a) analyzing a sample from said patient to obtain quantifying        data for one or more than one metabolite marker;    -   b) obtaining a ratio for each of the one or more than one        metabolite marker to an internal control metabolite;    -   c) comparing each ratio of said one or more than one metabolite        marker to the internal control metabolite to corresponding data        obtained from one or more than one reference sample, wherein        said comparison can be used to diagnose AO-PBD or the risk of        AO-PBD.

The step of analyzing the sample can be as described above. The one ormore than one reference sample may be a first reference sample obtainedfrom a non-AO-PBD control individual. The “internal control metabolite”refers to an endogenous metabolite naturally present in the patient. Anysuitable endogenous metabolite that does not vary over the diseasestates can be used as the internal control metabolite. For example, andwithout wishing to be limiting, the internal control metabolite may bephosphatidylethanolamine 16:0/18:0 (PtdEtn 16:0/18:0, M01), as shown inTable 5; this internal control metabolite has a molecular formula ofC₃₉H₇₈NO₈P, and a structure characterized as

Use of the ratio of the metabolite marker to the internal controlmetabolite offers measurement that are more stable and reproducible thanmeasurement of absolute levels of the metabolite marker. As the internalcontrol metabolite is naturally present in all samples and does notappear to vary significantly over disease states, the sample-to-samplevariability (due to handling, extraction, etc) is minimized.

The molecules described in the invention are listed in Table 5. Thisselection of molecules represents a representative sampling of diacyl,plasmanyl, and plasmenyl GPEs. However, someone skilled in the art wouldrecognize that other molecules of similar structure which are involvedin similar biochemical pathways could be used for similar purposes asdescribed below. All such modifications of the invention arecontemplated herein.

The present invention also provides high throughput methods fordiagnosis of AO-PBD. The method involves fragmentation of the parentmolecule; in a non-limiting example, this may be accomplished by aQ-Trap™ system. Detection of the metabolites may be performed using oneof various assay platforms, including colorimetric chemical assays (UV,or other wavelength), antibody-based enzyme-linked immunosorbant assays(ELISAs), chip-based and polymerase-chain reaction for nucleic aciddetection assays, bead-based nucleic-acid detection methods, dipstickchemical assays or other chemical reaction, image analysis such asmagnetic resonance imaging (MRI), positron emission tomography (PET)scan, computerized tomography (CT) scan, nuclear magnetic resonance(NMR), and various mass spectrometry-based systems. The preferred methodis a high throughput screening assay.

High throughput screening (HTS) was performed with a linear ion trapmass spectrometer (Q-trap 4000, Applied Biosystem) coupled with Agilent1100 LC system. Sample was prepared by adding 15 uL of internal standard(5 μg/mL of (24-13C)-Cholic Acid in methanol) to 120 uL ethyl acetatefraction of each sample. 100 ul sample was injected by flow injectionanalysis (FIA), and monitored under negative APCI mode. The method wasbased on multiple reaction monitoring (MRM) scan mode of oneparent/daughter transition for each metabolite and one internalstandard. Each transition was scanned for 70 ms for a total cycle timeof 2.475 sec. The isocratic 10% EtOAc in MeOH elution was performed witha flow rate at 360 μl/min for 1 min. The source parameters were set asfollows: CUR: 10.0, CAD: 8, NC: −4.0, TEM: 400, GS1: 30, GS2: 50,interface heater on. The compound parameters were set as follows: DP:−120.0, EP: −10, NC: −4.0, CE: −40, CXP: −15. FIG. 23 illustrates arepresentative standard curve for this method for PlsEtn 16:0/22:6generated by diluting a normal serum sample while maintaining a constantconcentration of internal standard (24-13C)-Cholic Acid).

According to the present invention, there is a diagnostic relationshipbetween decreased plasmalogens and cancer and/or dementia. Sincesubjects with an age-related plasmalogen deficiency have an elevatedrisk of developing cancer or dementia, this invention also provides amethod of diagnosing an elevated risk of getting cancer or dementia in asubject by measuring the levels of one or more than one plasmenyl orplasmanyl ether lipid present in a serum sample taken from a subject ofunknown disease status and comparing the levels to “normal” orage-related deficiency reference levels and through this comparisonarriving at a determination of elevated risk or not. The samples and thediagnostic methods according to this aspect of the invention are asdescribed in detail above.

Since there is an increased prevalence of an age-related plasmalogendeficiency in subjects currently suffering from cancer or dementia, thepresent invention also discloses a novel method for identifying subjectswith undiagnosed cancer or dementia. Accordingly, the present inventiondiscloses a novel method of identifying undiagnosed cancer or dementiain one or more than one subject by measuring the levels of one or morethan one plasmenyl or plasmanyl ether lipid present in a serum sampletaken from a subject of unknown disease status and comparing theselevels to “normal” or age-related plasmalogen deficiency referencelevels. The samples and the diagnostic methods according to this aspectof the invention are as described in detail above. Subjects that testpositive for an age-related plasmalogen deficiency are then tested byconventional cancer and dementia diagnostic methods to determine thelocation of the cancer and/or the severity and type of dementia presentin said subject.

The utility of the present invention will be further illustrated usingthe following examples.

EXAMPLES Example 1 Characterization of AO-PBD as a Separate and DistinctDisease State

In order to determine if AO-PBD is truly a separate disease state ormerely a symptom of a previously characterized human disease we executedthe following experiments:

Co-Morbidity Analysis

Since the symptoms of RCDP implicate both cancer and neurologicaldisease, we investigated the levels of various GPEs in lung, breast,colon, prostate, ovarian, and renal cancers, the three types of multiplesclerosis (relapsing remitting, secondary progressive, and primaryprogressive), probable dementia of the Alzheimer's type, and inpathologically confirmed Alzheimer's. The serum levels of four diacyl,eight plasmanyl, and eight plasmenyl GPEs and free DHA and arachidonicacid were analyzed in 1369 subjects of various ages and diseases (Tables4 and 5). Tables 6 to 11 show the results for each of the moleculesstudied. FIGS. 2 and 3 display the average and SEM of two prototypicalplasmenyl GPEs (16:0/18:2 and 16:0/22:6 respectively). Plasmenyl16:0/18:2 is a prototypical white matter plasmalogen containing a simpledi-unsaturated fatty acid at sn-2 (linoleic acid) and plasmenyl16:0/22:6 is a prototypical gray matter plasmalogen containing apolyunsaturated fatty acid at sn-2 (DHA). Each molecule is expressed asthe ratio to the diacyl GPE 16:0/18:0 and further normalized to the meancontrol population ratio for ease of viewing. Plasmenyl 16:0/18:2 and16:0/22:6 are significantly lower in all of the cancers and in probableAlzheimer's, but not in any of the multiple sclerosis groups. In fact,plasmenyl 16:0/22:6 is actually statistically elevated in secondaryprogressive and primary progressive MS. The other important observationfrom these two graphs is that there is a significant decrease inplasmalogens in the age 60-69 age group versus the age 50-59 group.

Determination of Whether Decreased Levels are Due to Increased OxidativeDegradation of Plasmalogens

As is shown in FIGS. 1 and 22, the final step in plasmalogen synthesisoccurs in the ER and involves the desaturation of the 1-O-ether to the1-O-vinyl ether. This 1-O-vinyl ether is critical to many of theproperties of plasmalogens, especially their anti-oxidant capacity. Anysituation that results in an increase in oxidative stress remote fromthe synthesis would preferentially deplete the plasmenyl species. Toinvestigate this, the plasmanyl/plasmenyl pairs for 16:0/18:2 and16:0/22:6 were measured (FIG. 4). What we observed was that in thediseases that showed a decrease in serum plasmalogen levels, both theplasmanyl and plasmenyl species decreased together. This indicates thatthe decreased levels of plasmalogens in cancer and dementia are not dueto increased oxidative stress in these diseases.

Determination of Prevalence of AO-PBD in Different Age Groups andDiseases

To determine the prevalence of AO-PBD we first calculated the plasmenyl16:0/18:2 and plasmenyl 16:0/22:6 ratio to diacyl 16:0/18:0 for all ofthe subjects, then we divided each subject by the normal population meanand then log 2 normalized each value. The lowest of the two log 2 valuesfor each subject was then used to create the population histograms shownin FIGS. 5 to 13. A cut-off value of −1 was used, however any cut-valuethat yields the desired sensitivity and specificity could be used.Subjects with log 2 values less than −1 have serum plasmalogen levelsless than 50% of the population average. To put this in perspective, theRDCP cell lines used by Perichon¹¹ had plasmalogen levels ofapproximately 30% of controls. Using this cut-off it was observed thatthe prevalence in cancer ranged from just under 50% in prostate cancerto 100% in breast cancer (FIG. 14). The difference between dementiasubjects and cognitively confirmed normals was 39% vs 6%, respectively.

Since a comorbidity between cancer and dementia is not obvious¹², and acommon underlying biochemical abnormality between cancer and dementiahas never before been established. These data strongly indicate thatAO-PBD is not a symptom of cancer or dementia and must therefore have anetiology of its own.

Investigations into the Etiology of AO-PBD

Since the etiology of dementia has been extensively studied, wedetermined the effect of dementia severity using 324 subjects (176female, 148 male) aged 56 to 95, comprised of 68 cognitively confirmednon-demented subjects (Mini Mental State Examination (MMSE ≧28)) and 256subjects currently diagnosed as having dementia (Alzheimer's DiseasesAssessment Score, cognitive sub-set (ADAS-cog) 6-70, MMSE 0-26)).Subjects were grouped into one of four dementia severity cohorts basedupon either their MMSE score [≧28=Cognitively Normal] or their ADAS-cogscore [5-19=low cognitive impairment; 20-39=moderate; 40-70=severe].Mean serum levels of eight PlsEtn and free docosahexaenoic acid (DHA,22:6) were determined for each group (FIG. 15). All eight PlsEtn in alldementia subgroups were observed to be significantly reduced relative tocognitive controls (24 pair-wise comparisons, t-test p-values 2.6e-2 to2.0e-10, median=3.9e-5). Free DHA was significantly decreased only inmoderately and severely demented subjects (p<0.05).

The data in FIG. 15 indicate that decreased serum PlsEtn correlate withincreasing dementia. To investigate this concept in detail, we performeda linear regression analysis using the mean serum PlsEtn 16:0/22:6 level(normalized to CN) of each of the dementia cohorts and the averageADAS-cog score for each of these three cohorts (FIG. 16). A very highcorrelation was observed between the mean serum

PlsEtn 16:0/22:6 level and the mean ADAS-cog scores of the threedementia cohorts (r²=0.99). However, this linear decrease did notextrapolate back to the CN group (X vs. CN). Assuming a clinicaldementia progression of 7.5 ADAS-cog units per year this extrapolationpredicts that that PlsEtn 16:0/22:6 levels begin to decline at leastseven years before clinical cognitive impairment (ADAS-cog=15) isevident. These data are consistent with the recent findings of Amieva etal¹³ in which a nine year prodromal phase of dementia of the Alzheimer'stype was observed. Considering that the effects of biochemical changesare rarely linear, but more commonly reflect an exponential effect, theprodromal biochemical phase would be expected to be longer than theprodromal clinical phase. This is supported by the fact that amyloidplaques begin to accumulate in 40-49 year olds¹⁴ but Alzheimer's doesnot begin to clinically manifest until late 60's early 70's. Based uponthese two studies and our own evidence that serum plasmalogens decreasebefore clinical symptoms occur, the etiology of dementia can beexpressed according to FIG. 17.

Furthermore, most cancers are predicted to have a 10-15 year prodromalperiod. Assuming a 15 year prodromal period for cancer, the percentageof asymptomatic subjects that will get cancer in the next 15 years canbe calculated (FIG. 18). Similar calculations can be made for dementia.All values were calculated based upon Canadian cancer and dementiastatistics. FIG. 19 displays the actual prevalence of AO-PBD inasymptomatic population controls aged 50-59 and 60-69. These values arecompared to the percentage of asymptomatic subjects that will get eithercancer in 15 years or dementia in 20 years followed by the sum of thesetwo populations. The last column shows, based upon the prevalence ofAO-PBD in these diseases (40% for dementia and 70% for cancer), whatpercentage of these subjects would be expected to have contracted canceror dementia as a result of having a pre-existing disease of AO-PBD. Whatthis analysis revealed is that of 50-59 year-olds 20% would be expectedto get either cancer or dementia in the next 15-20 years and 40% of60-69 year-olds will get either cancer or dementia. These data areremarkably close to the observed prevalence of AO-PBD in 50-59 (15%) and60-69 year-olds (30%).

To be confident that, in dementia, that AO-PBD was not simply a resultof Alzheimer's Disease (AD) pathology, we investigated serum samplescollected from 20 subjects (10 male and 10 female), pathologicallyconfirmed to have AD and 19 subjects pathologically confirmed not tohave AD. As can be seen by FIGS. 20 and 21, AO-PBD is present in only55% of the AD cases and in 16% of the controls. This means that in 45%of AD subjects, the underlying cause is something other than AO-PBD.Clearly AO-PBD and AD are not the same disease.

What the above studies show is that AO-PBD shows a separate and distinctetiology from both cancer and dementia. Whereas the prevalence of cancerand dementia continue to increase with increasing age to at least age90, the prevalence of AO-PBD peaks at age 60-69 and then decreases fromage 70 onward. Furthermore although AO-PBD exhibits a similarbiochemical profile to RCDP, it should not be confused with RCDP. Thethree forms of RDCP are all genetic disorders that affect children.Although, the underlying cause of AO-PBD is at this time unknown, it iscertainly not an inborn error of metabolism. The relationship betweenRCDP and AO-PBD is similar to the relationship between Down's syndrome(a genetically determined disease) and Alzheimer's Disease (an adultonset disease of unknown cause). Both Down's syndrome and Alzheimer'sDisease exhibit similar biochemical features (i.e. accumulation ofamyloid plaques in the brain), but the clinical manifestations aredramatically different.

Example 2 Identification of Subjects that have AO-PBD Using Serum Levelsof Metabolites

Using a validated analytical method such as that described above inExample 1, the mean±SEM serum levels for all or a subset of all of themetabolites listed in Table 5 were measured for a plurality ofcognitively normal, cancer-free subjects. This can be done for males andfemales separately or combined. The mean value for each metabolite someasured becomes the normal reference value.

Using a validated analytical method such as that described above, theserum level of each or a subset of all of the metabolites listed inTable 5 for test subject were calculated.

The ratio of the serum level of the test subject to average serum levelof the normal population was then determined and this ratio was comparedto a cut-off value (for example, but not meant to be limiting, a valueof 0.5 was used throughout this application). Subjects with a ratio ofless than 0.5 are deemed to have AO-PBD.

Example 3 Identification of Subjects that have AO-PBD Using aMathematically Determined Plasmalogen Score of the Ratio of Serum Levelsof Metabolites Listed in Table 5 to an Endogenous Reference Metabolite

Using a validated analytical method such as that described above inExample 1, the mean±SEM serum levels for all or a subset of all of themetabolites listed in Table 5 for a plurality of cognitively normal,cancer-free subjects was determined. This can be done for males andfemales separately or combined.

The ratio of these metabolites to the corresponding average from thecognitively normal or known dementia cohort was determined. The data wastransformed to log 2, as described in Example 1. The lowest log 2 scoreof M16 and M19 was selected. The log 2 score was then compared to acut-off value (−1.0 is used in this application) to determine if asubject has AO-PBD.

Example 4 Identification of AO-PBD Subjects by Comparing the Ratio ofOne or More than One Metabolite to an Endogenous Reference Metabolitefrom a Test Subject to the Average Such Ratio from a Normal ReferencePopulation

In order to decrease patient to patient variability, an endogenousmetabolite that does not change significantly between the variablesbeing tested can be used. For example, M01 could be used as it does notchange significantly in AO-PBD. Using a validated analytical method suchas that described above, the ratio of all or a subset of all of themetabolites listed in Table 5 was calculated. The mean±SEM serum ratiolevels for each of these metabolites for a plurality of normal subjectswas also calculated. This can be done for males and females separatelyor combined.

The serum ratio levels of one or more than one of the metabolites listedin Table 5 for a subject of unknown AO-PBD status was also determined.The ratio of one or more than one these metabolites to the correspondingaverage normal concentration was calculated and compared to a cut-offvalue. This comparison was used to determine if said subject has AO-PBD.

All citations are hereby incorporated by reference.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

TABLE 1 Biochemical functions performed by peroxisomes 1.Etherphospholid biosynthesis 2. Fatty acid β-oxidation 3. Fatty acidα-oxidation 4. Glyoxylate detoxification 5. Biosynthesis of cholesteroland dolichol 6. Pipecolic acid degradation 7. Biosynthesis ofdecosahexaemoic acid 8. Hydrogen peroxide metabolism 9. Aminoacidmetabolism

TABLE 2 Peroxisomal disorders and their enzymatic basis 1.Cerebro-hepato-renal syndrome of Generalized Zellweger (ZS) 2. Neonataladrenoleukodystrophy Generalized (NALD) 3. Infantile Refsum disease(IRD) Generalized 4. Hyperpipecolic acidemia (HPA) Generalized 5. RCDPType 1 (PTS2-receptor defect) Multiple 6. RCDP Type 2(DHAPAT-deficiency) DHAPAT 7. RCDP Type 3 (AlkylDHAP-synthaseAlkylDHAP-synthase deficiency) 8. Zellweger-like syndrome Multiple 9.X-linked adrenoleukodystrophy ALDp (XALD) 10. Acyl-CoA oxidasedeficiency Acyl-CoA oxidase (pseudo-NALD) 11. Bifunctional proteindeficiency D-Bifunctional protein 12. Peroxisomal thiolase (I)deficiency Thiolase (peroxisomal) 13. Hyperoxaluria Type 1 (alanine AGTglyoxylate aminotransferase deficiency) 14. Refsum disease (classicform) Phytanoyl-CoA (phytanoyl-CoA hydroxylase deficiency) hydroxylase15. Glutaric aciduria Type 3 Glutaryl-CoA oxidase 16. Mevalonate kinasedeficiency Mevalonate kinase 17. Acetalasaemia Catalase Abbreviationsused: DHAPAT = DiHydroxyAcetonePhosphate AcylTransferase; AGT = AlanineGlyoxylate aminoTransferase; PhyH = Phytanoyl-CoA Hydroxylane; ALDp =AdrenoLeukoDystrophy protein; RCDP = Rhizomelic ChondroDysplasiaPunctata.

TABLE 3 Differentiation of peroxisomal disorders: Biogenesis vs. singleenzyme deficiencies. Group 1 (Biogenosis defects) Zellweger syndromeNeonatal adrenoleukodystrophy Infantile Refsum disease Hyperpipecolicacidemia Rhizomelic chondrodyaplasia punctata (RCDP) Type 1Zellweger-like syndrome* Group 2 (Single Enzyme (protein) deficiencies):X-linked adrenoleukodystrophy Acyl-CoA oxidase deficiency Bifunctionalprotein deficiency Peroxisomal thiolase deficiency Rhizomelicchondrodysplasia punctata Type 2 (DHAPAT-deficiency) Rhizomelicchondrodysplasia punctata Type 3 (alkylDHAP synthase deficiency) Refsumdisease (Classic type) (phytanoyl-CoA hydroxylate deficiency) Glutamicaciduria Type 3 (glutaryl-CoA oxidase deficiency) Mevalonate kinasedeficiency Hyperoxaluria Type 1 (alanine glyoxylate aminotransferasedeficiency) Acetalassemia *Not established definitively

TABLE 4 Clinical Data Summary Disease Age (Mean) Age (SEM) Male Female nControl, All 56.6 0.6 184 253 437 Control, 20-39 33.9 0.9 12 19 31Control, 40-49 44.8 0.4 29 45 74 Control, 50-59 54.1 0.2 64 107 171Control, 60-69 64.0 0.3 44 55 99 Control, 70-95 77.3 0.7 35 27 62Cognitive Normal 77.2 0.8 32 36 68 Alzheimer's Disease 80.3 0.5 79 114194 Relapsing/Remitting 47.6 0.6 54 231 285 Secondary 51.3 2.0 8 16 24Progressive Primary Progressive 54.5 1.8 5 11 16 Colon Cancer 60.3 1.0131 89 220 Ovarian Cancer 60.7 2.9 0 20 20 Prostate Cancer 63.1 1.9 25 025 Lung Cancer 61.2 2.6 11 14 25 Renal Cancer 67.6 2.2 17 13 30 BreastCancer 57.5 2.6 0 25 25

TABLE 5 Table of Diacyl, Plasmanyl, and Plasmenyl GPEs and Free FattyAcids Measured in Serum. Metabolite Code Metabolite Name MolecularFormula Parent Mass M-H Mass Diagnostic Fragment Mass MS/MS TransitionM01 PtdEt 16:0/18:0 C39H78N1O8P1 719.54648 718.5 R1 (C16H31O2)-255718.0/255.0 M02 PtdEt 16:0/18:1 C39H76N1O8P1 717.53083 716.5 R1(C16H31O2)-255 716.0/255.0 M03 PtdEt 18:0/18:0 C41H82N1O8P1 747.57777746.5 R1 (C18H35O2)-283 746.0/283.0 M04 PtdEt 18:0/18:1 C41H80N1O8P1745.56213 744.5 R1 (C18H35O2)-283 744.0/283.0 M05 Plasmanyl 16:0/18:1C39H78N1O7P1 703.55156 702.5 R2 (C18H33O2)-281 702.0/281.0 M06 Plasmanyl16:0/18:2 C39H76N1O7P1 701.53591 700.5 R2 (C18H31O2)-279 700.0/279.0 M07Plasmanyl 16:0/20:4 C41H76N1O7P1 725.53591 724.5 R2 (C20H31O2)-303724.0/303.0 M08 Plasmanyl 16:0/22:4 C43H80N1O7P1 753.56721 752.5 R2(C22H35O2)-331 752.0/331.0 M09 Plasmanyl 16:0/22:6 C43H76N1O7P1749.53591 748.5 R2 (C22H31O2)-327 748.0/327.0 M10 Plasmanyl 18:0/18:1C41H82N1O7P1 731.58286 730.5 R2 (C18H33O2)-281 730.0/281.0 M11 Plasmanyl18:0/18:2 C41H80N1O7P1 729.56721 728.5 R2 (C18H31O2)-279 728.0/279.0 M12Plasmanyl 18:0/20:4 C43H80N1O7P1 753.56721 752.5 R2 (C20H31O2)-303752.0/303.0 M13 Plasmanyl 18:0/22:4 C45H84N1O7P1 781.59851 780.5 R2(C22H35O2)-331 780.0/331.0 M14 Plasmanyl 18:0/22:6 C45H80N1O7P1777.56721 776.5 R2 (C22H31O2)-327 776.0/327.0 M15 Plasmenyl 16:0/18:1C39H76N1O7P1 701.53591 700.5 R2 (C18H33O2)-281 700.0/281.0 M16 Plasmenyl16:0/18:2 C39H74N1O7P1 699.52026 698.5 R2 (C18H31O2)-279 698.0/279.0 M17Plasmenyl 16:0/20:4 C41H74N1O7P1 723.52026 722.5 R2 (C20H31O2)-303722.0/303.0 M18 Plasmenyl 16:0/22:4 C43H78N1O7P1 751.55156 750.5 R2(C22H35O2)-331 750.0/331.0 M19 Plasmenyl 16:0/22:6 C43H74N1O7P1747.52026 746.5 R2 (C22H31O2)-327 746.0/327.0 M20 Plasmenyl 18:0/18:1C41H80N1O7P1 729.56721 728.5 R2 (C18H33O2)-281 728.0/281.0 M21 Plasmenyl18:0/18:2 C41H78N1O7P1 727.55156 726.5 R2 (C18H31O2)-279 726.0/279.0 M22Plasmenyl 18:0/20:4 C43H78N1O7P1 751.55156 750.5 R2 (C20H31O2)- 303750.6/303.2 M23 Plasmenyl 18:0/22:4 C45H82N1O7P1 779.58286 778.5 R2(C22H35O2)-331 778.0/331.0 M24 Plasmenyl 18:0/22:6 C45H78N1O7P1775.55156 774.5 R2 (C22H31O2)-327 774.0/327.0 M25 Free 22:6 C22H32O2328.24022 327.2 (C21H31)-283 327.2/283.0 M26 Free 20:4 C20H32O2304.24022 303.2 (C19H31)-259 303.2/259.5

TABLE 6 Table of serum changes (relative to controls) for diacyl GPEs.Diacyl GPEs Raw IS 16:0/18:0 16:0/18:1 18:0/18:0 18:0/18:1 Ratio Data718.0/255.0 716.0/255.0 746.0/283.0 744.0/283.0 AD, All 1.01 0.97 0.801.02 7.1E−01 6.2E−01 2.9E−06 7.7E−01 RR, All 0.74 0.91 0.82 0.93 3.2E−185.4E−02 3.3E−07 1.5E−01 RR, 20-29 0.86 1.17 0.82 1.08 6.8E−01 8.6E−023.0E−01 3.5E−01 RR, 30-39 0.81 1.13 0.84 0.91 4.8E−02 4.7E−01 1.5E−016.2E−01 RR, 40-49 0.66 0.68 0.76 0.73 2.0E−09 9.3E−05 5.0E−04 2.7E−03RR, 50-59 0.82 0.99 0.86 1.01 1.4E−03 9.2E−01 1.8E−02 9.1E−01 RR, 60-690.70 0.87 0.87 0.92 3.4E−04 3.2E−01 2.0E−01 5.8E−01 Secondary 1.01 1.331.37 1.52 Progressive 8.9E−01 1.2E−02 7.3E−04 3.6E−04 Primary 0.88 0.971.08 1.00 Progressive 2.9E−01 8.4E−01 5.3E−01 9.9E−01 Colon Cancer, 1.130.99 0.92 0.69 All 2.7E−03 7.9E−01 5.5E−02 2.1E−09 Ovarian Cancer 1.090.87 0.89 0.73 3.7E−01 3.6E−01 3.3E−01 7.3E−02 Prostate Cancer 0.93 0.730.73 0.59 4.2E−01 3.1E−02 8.9E−03 2.8E−03 Lung Cancer 1.33 1.03 0.760.79 9.2E−04 8.1E−01 2.7E−02 1.6E−01 Renal Cancer 1.62 1.43 1.12 1.367.1E−13 2.8E−04 2.1E−01 6.0E−03 Breast Cancer 1.17 0.90 0.71 0.606.1E−02 4.2E−01 6.1E−03 3.3E−03

TABLE 7 Table of serum changes (relative to controls) for plasmanylGPEs. Plasmanyl GPEs 16:0/18:1 16:0/18:2 16:0/20:4 16:0/22:4 Raw IS702.0/ 700.0/ 724.0/ 752.0/ 16:0/22:6 18:0/18:1 18:0/18:2 18:0/20:418:0/22:4 18:0/22:6 Ratio Data 281.0 279.0 303.0 331.0 748.0/327.0730.0/281.0 728.0/279.0 752.0/303.0 780.0/331.0 776.0/327.0 AD, All 0.860.85 0.66 0.70 0.68 0.94 0.85 0.75 0.82 0.86 7.1E−04 3.0E−04 4.2E−094.5E−07 1.5E−06 1.1E−01 6.7E−05 2.3E−09 7.2E−06 2.0E−02 RR, All 0.810.88 0.79 0.84 0.81 0.90 0.94 0.87 0.89 0.91 2.8E−08 2.9E−04 3.1E−051.3E−03 2.7E−04 1.8E−03 7.4E−02 1.7E−04 1.3E−03 5.8E−02 RR, 20-29 0.710.80 0.68 0.63 0.79 0.90 1.07 0.83 0.77 1.15 2.7E−01 5.8E−01 9.0E−023.9E−01 3.1E−01 4.5E−01 5.8E−02 5.0E−01 8.9E−01 2.6E−01 RR, 30-39 0.730.71 0.67 0.71 0.83 0.91 0.77 0.80 0.86 1.00 9.1E−03 6.2E−03 8.0E−031.6E−02 2.6E−01 4.3E−01 3.3E−02 5.4E−02 1.6E−01 1.0E+00 RR, 40-49 0.720.78 0.74 0.70 0.74 0.77 0.83 0.80 0.77 0.85 1.7E−05 8.2E−04 1.6E−021.6E−03 2.9E−02 1.3E−04 6.8E−03 3.5E−03 1.1E−04 1.3E−01 RR, 50-59 0.850.89 0.76 0.83 0.79 0.96 0.99 0.87 0.91 0.94 6.7E−03 5.2E−02 1.2E−032.1E−02 8.8E−03 4.8E−01 8.0E−01 2.5E−02 9.7E−02 3.9E−01 RR, 60-69 0.770.93 0.84 0.85 0.94 0.85 0.92 0.91 0.94 1.10 3.1E−02 4.7E−01 3.0E−013.4E−01 7.2E−01 1.0E−01 3.5E−01 4.0E−01 6.0E−01 4.9E−01 Secondary 1.271.36 1.49 1.35 1.77 1.31 1.35 1.36 1.36 1.76 Progressive 1.1E−02 6.7E−041.6E−03 2.2E−02 1.9E−05 1.1E−03 1.7E−04 5.7E−04 3.1E−04 8.0E−08 Primary0.94 1.09 1.09 1.00 1.24 1.01 1.05 1.10 1.10 1.29 Progressive 6.1E−015.0E−01 6.2E−01 9.9E−01 2.3E−01 9.2E−01 6.4E−01 4.1E−01 4.2E−01 7.9E−02Colon 0.80 0.61 0.67 0.62 0.69 0.89 0.63 0.78 0.84 0.99 Cancer, All7.7E−06 1.5E−20 2.2E−08 2.2E−12 1.5E−07 4.8E−03 2.6E−24 1.5E−08 2.5E−058.1E−01 Ovarian Cancer 0.82 0.70 0.63 0.59 0.70 0.94 0.74 0.76 0.78 1.131.1E−01 9.3E−03 2.2E−02 1.1E−02 9.0E−02 5.6E−01 8.2E−03 3.2E−02 4.5E−023.8E−01 Prostate Cancer 0.68 0.63 0.49 0.63 0.55 0.79 0.70 0.67 0.820.72 1.8E−03 3.4E−04 3.6E−04 1.1E−02 5.1E−03 1.9E−02 6.9E−04 7.4E−046.1E−02 3.4E−02 Lung Cancer 0.66 0.55 0.35 0.36 0.29 0.89 0.69 0.62 0.730.55 1.4E−03 1.2E−05 6.5E−06 1.6E−05 8.5E−06 2.6E−01 8.6E−04 1.8E−046.4E−03 7.8E−04 Renal Cancer 1.15 0.97 0.72 0.91 0.85 1.29 1.05 0.911.08 1.24 1.2E−01 7.1E−01 3.5E−02 5.1E−01 3.0E−01 9.9E−04 5.5E−013.4E−01 3.9E−01 4.8E−02 Breast Cancer 0.67 0.55 0.39 0.45 0.36 0.87 0.640.63 0.79 0.51 1.3E−03 8.8E−06 2.6E−05 1.6E−04 6.8E−05 1.7E−01 4.8E−052.2E−04 3.2E−02 1.9E−04

TABLE 8 Table of serum changes (relative to controls) for plasmenylGPEs. Plasmenyl Pes 16:0/18:1 16:0/18:2 16:0/20:4 16:0/22:4 Raw IS700.0/ 698.0/ 722.0/ 750.0/ 16:0/22:6 18:0/18:1 18:0/18:2 18:0/20:418:0/22:4 18:0/22:6 Ratio Data 281.0 279.0 303.0 331.0 746.0/327.0728.0/281.0 726.0/279.0 750.6/303.2 778.0/331.0 774.0/327.0 AD, All 0.820.78 0.66 0.70 0.72 0.85 0.80 0.76 0.74 0.80 7.5E−07 4.7E−08 4.0E−092.6E−08 7.6E−06 3.3E−04 7.0E−06 1.7E−05 6.5E−08 1.5E−03 RR, All 0.640.88 0.80 0.85 0.82 0.84 0.90 0.81 0.83 0.85 1.0E−07 4.4E−04 1.3E−041.1E−03 6.5E−04 6.1E−06 6.5E−03 1.6E−04 4.2E−05 4.4E−03 RR, 20-29 0.770.81 0.60 0.62 0.78 0.69 0.82 0.62 0.67 0.71 3.8E−01 5.1E−01 7.2E−023.5E−01 2.9E−01 1.5E−01 4.5E−01 4.8E−02 2.5E−01 1.1E−01 RR, 30-39 0.760.71 0.70 0.73 0.85 0.70 0.70 0.70 0.72 0.86 1.2E−02 4.4E−03 3.8E−023.4E−02 3.7E−01 9.1E−03 1.2E−02 4.0E−02 1.5E−02 4.2E−01 RR, 40-49 0.730.82 0.77 0.72 0.81 0.75 0.80 0.78 0.74 0.81 4.3E−06 4.5E−03 3.2E−029.6E−04 8.7E−02 1.7E−04 5.4E−03 2.1E−02 2.0E−04 8.4E−02 RR, 50-59 0.900.91 0.79 0.84 0.82 0.90 0.93 0.80 0.83 0.86 5.2E−02 1.3E−01 1.2E−023.0E−02 2.8E−02 1.0E−01 2.8E−01 1.5E−02 7.5E−03 1.2E−01 RR, 60-69 0.840.90 0.85 0.90 0.94 0.87 0.95 0.90 0.87 1.03 7.0E−02 3.2E−01 3.1E−015.5E−01 7.0E−01 2.4E−01 6.5E−01 4.7E−01 4.4E−01 8.5E−01 Secondary 1.151.27 1.36 1.47 1.69 1.25 1.33 1.46 1.41 1.77 Progressive 1.1E−01 1.0E−021.8E−02 1.1E−03 4.0E−05 2.0E−02 5.4E−03 1.7E−03 8.7E−04 3.0E−06 Primay0.94 1.04 1.11 1.03 1.29 1.03 1.11 1.18 1.09 1.46 Progressive 6.2E−017.4E−01 5.7E−01 8.7E−01 1.4E−01 8.4E−01 4.3E−01 3.1E−01 5.6E−01 1.7E−02Colon 0.94 0.62 0.62 0.61 0.75 0.83 0.56 0.59 0.66 0.75 Cancer, All1.6E−01 1.5E−20 6.3E−11 1.6E−14 3.0E−05 1.5E−04 1.7E−23 1.2E−13 2.1E−141.6E−05 Ovarian Cancer 0.89 0.68 0.68 0.66 0.82 0.82 0.65 0.69 0.68 0.872.8E−01 4.6E−03 5.8E−02 2.4E−02 3.1E−01 1.2E−01 5.8E−03 4.9E−02 1.7E−024.5E−01 Prostate Cancer 0.74 0.62 0.49 0.66 0.66 0.64 0.57 0.53 0.710.63 3.5E−03 1.7E−04 6.2E−04 1.3E−02 2.7E−02 5.9E−04 1.2E−04 7.8E−041.5E−02 1.5E−02 Lung Cancer 0.82 0.66 0.38 0.42 0.36 0.65 0.57 0.45 0.550.39 5.4E−02 8.8E−04 3.2E−05 2.8E−05 4.3E−05 1.2E−03 1.3E−04 8.6E−051.6E−04 7.2E−05 Renal Cancer 1.08 0.88 0.75 0.90 0.94 1.03 0.81 0.800.92 0.96 3.5E−01 2.1E−01 6.2E−02 4.2E−01 6.6E−01 7.7E−01 6.1E−021.3E−01 4.9E−01 7.6E−01 Breast Cancer 0.77 0.57 0.39 0.47 0.39 0.65 0.530.43 0.58 0.40 1.1E−02 1.8E−05 4.4E−05 1.0E−04 9.1E−05 1.0E−03 2.5E−055.3E−05 4.1E−04 9.7E−05

TABLE 9 Table of serum changes (relative to controls) of diacyl GPEs,DHA and AA using diacyl 16:0/18:0 GPE as an internal standard. DiacylGPEs DHA AA 16:0/18:0 16:0/18:1 18:0/18:0 18:0/18:1 22:6 20:4718.0/255.0 716.0/255.0 746.0/283.0 744.0/283.0 327.2/283.0 303.2/259.5AD, All 1.00 0.89 0.76 0.93 0.70 0.71 #DIV/0! 3.3E−04 7.4E−19 9.0E−022.1E−10 8.0E−12 RR, All 1.00 1.18 1.09 1.22 0.96 1.05 #DIV/0! 1.1E−093.9E−04 3.1E−08 3.2E−01 2.8E−01 RR, 20-29 1.00 1.19 0.94 1.14 0.75 0.83#DIV/0! 1.1E−02 2.3E−01 1.3E−01 4.2E−02 7.5E−02 RR, 30-39 1.00 1.27 1.011.03 1.23 0.97 #DIV/0! 1.0E−02 8.8E−01 8.1E−01 1.9E−01 8.2E−01 RR, 40-491.00 1.01 1.12 1.07 1.35 1.38 #DIV/0! 8.5E−01 2.2E−02 3.2E−01 6.7E−045.3E−04 RR, 50-59 1.00 1.18 1.03 1.23 0.80 0.88 #DIV/0! 4.3E−04 5.1E−015.5E−04 4.1E−03 5.9E−02 RR, 60-69 1.00 1.22 1.21 1.31 1.04 1.02 #DIV/0!5.3E−03 5.3E−03 5.0E−03 7.7E−01 8.4E−01 Secondary Progressive 1.00 1.271.32 1.43 0.92 0.90 #DIV/0! 6.9E−04 1.5E−05 2.7E−05 5.2E−01 3.9E−01Primary Progressive 1.00 1.13 1.25 1.14 0.91 0.91 #DIV/0! 1.7E−015.0E−03 2.5E−01 5.4E−01 5.2E−01 Colon Cancer, All 1.00 0.88 0.82 0.631.15 0.82 #DIV/0! 9.2E−05 1.2E−11 1.1E−23 3.2E−03 2.3E−05 Ovarian Cancer1.00 0.81 0.80 0.67 0.96 0.75 #DIV/0! 2.2E−02 9.9E−03 2.3E−03 7.4E−014.2E−02 Prostate Cancer 1.00 0.79 0.79 0.66 0.97 0.87 #DIV/0! 5.0E−033.2E−03 4.3E−04 8.3E−01 2.5E−01 Lung Cancer 1.00 0.68 0.55 0.47 0.800.87 #DIV/0! 2.2E−05 1.4E−10 7.0E−08 9.6E−02 2.3E−01 Renal Cancer 1.000.88 0.68 0.81 0.39 0.36 #DIV/0! 7.5E−02 4.4E−07 3.0E−02 2.4E−08 4.0E−10Breast Cancer 1.00 0.73 0.58 0.48 0.68 0.82 #DIV/0! 2.6E−04 1.6E−097.8E−08 6.5E−03 1.0E−01

TABLE 10 Table of serum changes (relative to controls) of plasmanylGPEs, using diacyl 16:0/18:0 GPE as an internal standard. Plasmanyl GPEs16:0/18:1 16:0/18:2 16:0/20:4 16:0/22:4 702.0/ 700.0/ 724.0/ 752.0/16:0/22:6 18:0/18:1 18:0/18:2 18:0/20:4 18:0/22:4 18:0/22:6 281.0 279.0303.0 331.0 748.0/327.0 730.0/281.0 728.0/279.0 752.0/303.0 780.0/331.0776.0/327.0 AD, All 0.84 0.83 0.62 0.66 0.63 0.91 0.82 0.71 0.79 0.805.6E−08 1.2E−06 4.7E−16 3.8E−13 1.5E−13 5.8E−05 8.0E−10 2.6E−20 8.1E−133.7E−07 RR, All 1.09 1.16 1.05 1.11 1.07 1.20 1.23 1.15 1.17 1.191.2E−03 7.0E−07 3.2E−01 1.3E−02 1.5E−01 2.5E−21 1.7E−16 8.7E−07 8.2E−101.2E−06 RR, 20-29 0.88 1.02 0.84 0.75 0.87 1.04 1.21 0.98 0.92 1.155.0E−01 9.1E−01 8.8E−02 4.2E−01 2.0E−01 3.0E−02 9.0E−03 5.6E−01 7.4E−015.4E−01 RR, 30-39 0.91 0.88 0.79 0.84 1.01 1.13 0.95 0.98 1.03 1.232.0E−01 1.9E−01 4.7E−02 9.2E−02 9.7E−01 2.4E−02 5.2E−01 8.4E−01 6.6E−018.2E−02 RR, 40-49 1.05 1.12 1.05 0.99 1.09 1.13 1.19 1.16 1.11 1.254.2E−01 6.9E−02 6.3E−01 9.3E−01 3.8E−01 2.2E−03 7.9E−04 1.8E−02 4.7E−029.0E−04 RR, 50-59 1.03 1.07 0.91 1.01 0.95 1.16 1.17 1.04 1.07 1.105.2E−01 1.5E−01 2.2E−01 8.7E−01 4.5E−01 1.8E−06 9.0E−05 4.2E−01 8.9E−028.5E−02 RR, 60-69 1.09 1.28 1.13 1.16 1.29 1.20 1.27 1.23 1.28 1.522.5E−01 6.0E−03 3.5E−01 2.2E−01 4.1E−02 8.6E−04 8.8E−04 9.9E−03 1.4E−034.7E−05 Secondary 1.22 1.29 1.40 1.28 1.67 1.25 1.27 1.28 1.28 1.68Progressive 3.6E−03 2.1E−03 2.0E−03 2.5E−02 3.9E−06 5.9E−06 4.9E−045.8E−04 2.9E−04 4.6E−10 Primary 1.10 1.25 1.28 1.13 1.48 1.15 1.19 1.221.19 1.46 Progressive 2.8E−01 2.9E−02 7.2E−02 3.8E−01 4.3E−03 2.3E−024.4E−02 3.0E−02 4.6E−02 3.2E−04 Colon 0.70 0.53 0.58 0.55 0.61 0.78 0.550.69 0.76 0.86 Cancer, All 5.6E−24 1.1E−39 2.4E−18 1.6E−22 1.2E−155.8E−25 3.3E−51 3.8E−24 1.9E−16 1.4E−04 Ovarian Cancer 0.74 0.61 0.570.53 0.63 0.85 0.65 0.68 0.70 1.00 1.3E−03 1.7E−04 1.9E−03 6.1E−041.1E−02 1.1E−02 3.2E−05 3.2E−04 3.4E−04 9.9E−01 Prostate Cancer 0.750.68 0.53 0.67 0.62 0.85 0.73 0.71 0.87 0.80 6.5E−04 6.0E−04 1.3E−046.2E−03 3.5E−03 4.6E−03 4.0E−04 2.6E−04 7.7E−02 4.3E−02 Lung Cancer 0.450.37 0.24 0.24 0.20 0.60 0.47 0.44 0.51 0.37 1.2E−13 9.0E−12 1.1E−095.1E−10 1.2E−09 1.4E−13 2.2E−12 6.3E−12 1.1E−10 6.6E−10 Renal Cancer0.68 0.56 0.42 0.53 0.49 0.76 0.61 0.54 0.63 0.74 1.2E−06 1.3E−072.8E−07 2.3E−05 2.0E−05 7.6E−07 1.6E−08 5.9E−10 1.0E−07 4.6E−03 BreastCancer 0.54 0.43 0.29 0.33 0.27 0.72 0.51 0.51 0.64 0.41 4.7E−10 6.2E−101.2E−08 4.9E−08 2.6E−08 1.6E−07 1.5E−10 7.8E−10 1.5E−06 8.0E−09

TABLE 11 Table of serum changes (relative to controls) of plasmenylGPEs, using diacyl 16:0/18:0 GPE as an internal standard. Plasmenyl GPEs16:0/18:1 16:0/18:2 16:0/20:4 16:0/22:4 700.0/ 698.0/ 722.0/ 750.0/16:0/22:6 18:0/18:1 18:0/18:2 18:0/20:4 18:0/22:4 18:0/22:6 281.0 279.0303.0 331.0 746.0/327.0 728.0/281.0 726.0/279.0 750.6/303.2 778.0/331.0774.0/327.0 AD, All 0.79 0.76 0.63 0.65 0.67 0.83 0.78 0.73 0.71 0.762.9E−16 1.1E−11 3.5E−15 2.5E−16 6.0E−11 1.1E−07 7.0E−09 2.5E−09 2.1E−151.8E−06 RR, All 1.12 1.17 1.08 1.12 1.09 1.12 1.18 1.10 1.10 1.128.5E−07 1.3E−07 1.2E−01 3.1E−03 7.0E−02 1.3E−04 3.3E−07 3.0E−02 3.5E−031.4E−02 RR, 20-29 0.94 1.00 0.75 0.74 0.85 0.84 0.99 0.79 0.82 0.807.0E−01 9.1E−01 6.0E−02 3.8E−01 1.7E−01 2.1E−01 7.3E−01 4.7E−02 3.0E−016.3E−02 RR, 30-39 0.95 0.88 0.85 0.89 1.04 0.89 0.87 0.87 0.88 1.073.9E−01 1.9E−01 2.3E−01 2.4E−01 7.9E−01 1.7E−01 2.1E−01 3.2E−01 1.6E−016.8E−01 RR, 40-49 1.10 1.18 1.12 1.03 1.18 1.09 1.14 1.13 1.04 1.175.3E−02 7.1E−03 2.7E−01 7.1E−01 8.3E−02 1.5E−01 4.9E−02 1.8E−01 5.7E−019.4E−02 RR, 50-59 1.07 1.09 0.96 1.02 0.98 1.08 1.11 0.98 1.00 1.027.6E−02 7.4E−02 6.2E−01 7.6E−01 7.7E−01 1.0E−01 4.1E−02 8.0E−01 1.0E+007.6E−01 RR, 60-69 1.17 1.26 1.17 1.22 1.33 1.21 1.31 1.24 1.20 1.441.2E−02 6.5E−03 2.1E−01 8.1E−02 2.4E−02 1.3E−02 3.2E−03 5.2E−02 8.6E−021.8E−03 Secondary 1.10 1.21 1.33 1.41 1.63 1.21 1.27 1.42 1.35 1.71Progressive 1.2E−01 2.2E−02 9.3E−03 2.7E−04 6.8E−06 9.8E−03 6.9E−034.5E−04 2.9E−04 3.4E−07 Primary 1.08 1.19 1.29 1.15 1.51 1.18 1.28 1.361.20 1.70 Progressive 3.2E−01 9.2E−02 6.0E−02 2.7E−01 2.2E−03 7.7E−022.5E−02 1.3E−02 8.5E−02 3.6E−05 Colon 0.84 0.55 0.55 0.57 0.68 0.72 0.490.53 0.61 0.67 Cancer, All 3.8E−10 3.3E−38 1.7E−22 1.6E−24 1.8E−111.6E−18 4.7E−42 2.0E−27 3.5E−27 5.6E−12 Ovarian Cancer 0.80 0.60 0.620.59 0.74 0.74 0.58 0.63 0.61 0.77 4.4E−03 5.1E−05 5.4E−03 8.4E−047.1E−02 3.1E−03 9.1E−05 3.7E−03 1.6E−04 1.2E−01 Prostate Cancer 0.780.66 0.54 0.69 0.72 0.70 0.62 0.58 0.75 0.70 6.2E−04 1.4E−04 1.2E−044.8E−03 3.2E−02 1.5E−04 6.6E−05 2.3E−04 6.9E−03 2.0E−02 Lung Cancer 0.610.50 0.29 0.29 0.27 0.47 0.41 0.33 0.39 0.29 1.1E−09 1.3E−08 3.9E−097.7E−11 2.0E−08 3.8E−11 1.0E−09 5.8E−09 4.2E−11 4.2E−08 Renal Cancer0.65 0.52 0.44 0.53 0.55 0.61 0.48 0.48 0.54 0.56 1.2E−09 3.7E−094.0E−07 2.8E−06 1.4E−04 8.4E−08 2.6E−09 7.1E−07 5.2E−08 2.2E−04 BreastCancer 0.62 0.46 0.31 0.36 0.30 0.53 0.43 0.35 0.46 0.32 2.3E−09 1.1E−091.0E−08 5.0E−09 7.1E−08 3.7E−09 2.2E−09 1.4E−08 4.5E−09 1.3E−07

REFERENCES

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1-7. (canceled)
 8. A method of diagnosing adult onset plasmalogenbiosynthesis disorder, diagnosing a plasmalogen deficiency-related humanhealth disorder, or the risk of a plasmalogen deficiency-related humanhealth disorder in a subject, comprising the steps of: a) analyzing asample from said subject to obtain quantifying data for one or more thanone metabolite markers; b) comparing said quantifying data for said oneor more than one metabolite marker to corresponding data obtained fromone or more than one reference sample to identify a decrease in thelevel of said one or more than one metabolite marker in said sample; andc) using said decrease in level of said one or more than one metabolitemarker in said sample for diagnosing the presence of adult onsetplasmalogen biosynthesis disorder in said subject, wherein the one ormore than one metabolite marker comprises one or more than one moleculeselected from the group consisting of: diacylglycerylphosphoethanolamines, plasmanyl glycerylphosphoethanolamines,plasmenyl glycerylphosphoethanolamines, and free fatty acids.
 9. Themethod according to claim 8, further comprising: analyzing a sample fromsaid subject to obtain quantifying data for one or more than oneinternal control metabolite; and obtaining a ratio for each of thelevels of said one or more than one metabolite marker to the levelobtained for the one or more than one internal control metabolite;wherein the comparing step (b) comprises comparing each ratio to one ormore corresponding ratios obtained for the one or more than onereference sample.
 10. The method according to claim 9, wherein thediacyl glycerylphosphoethanolamines are selected from the groupconsisting of: PtdEt 16:0/18:0, PtdEt 16:0/18:1, PtdEt 18:0/18:0, PtdEt18:0/18:1, and combinations thereof.
 11. The method according to claim9, wherein the plasmanyl glycerylphosphoethanolamines are selected fromthe group consisting of: plasmanyl 16:0/18:1, plasmanyl 16:0/18:2,plasmanyl 16:0/20:4, plasmanyl 16:0/22:4, plasmanyl 16:0/22:6, plasmanyl18:0/18:1, plasmanyl 18:0/18:2, plasmanyl 18:0/20:4, plasmanyl18:0/22:4, plasmanyl 18:0/22:6, and combinations thereof.
 12. The methodaccording to claim 9, wherein the plasmenyl glycerylphosphoethanolaminesis selected from the group consisting of: plasmenyl 16:0/18:1, plasmenyl16:0/18:2, plasmenyl 16:0/20:4, plasmenyl 16:0/22:4, plasmenyl16:0/22:6, plasmenyl 18:0/18:1, plasmenyl 18:0/18:2, plasmenyl18:0/20:4, plasmenyl 18:0/22:4, plasmenyl 18:0/22:6, and combinationsthereof.
 13. The method according to claim 9, wherein the free fattyacid is selected from the group consisting of: free docosohexanoic acid(DHA) 22.6, free DHA 20:4 and combinations thereof.
 14. The methodaccording to claim 8, wherein the plasmalogen deficiency-relateddisorder is selected from the group consisting of: breast cancer,colorectal cancer, prostate cancer, lung cancer, ovarian cancer, renalcancer, Alzheimer's Disease, Dementia, or Cognitive impairment.
 15. Themethod according to claim 14, wherein the subject is older than fortyyears of age.
 16. The method according to claim 8, wherein thequantifying data is obtained using a Fourier transform ion cyclotronresonance, time of flight, orbitrap, quadrupole or triple quadrupolemass spectrometer.
 17. The method according to claim 16, wherein themass spectrometer is equipped with a chromatographic system.
 18. Themethod according to claim 8, wherein the sample is a blood sample. 19.The method according to claim 8, wherein the sample is a blood serumsample.
 20. The method according to claim 8, wherein the sample is acerebral spinal fluid sample.
 21. The method according to claim 8,wherein a liquid/liquid extraction is performed on the sample wherebynon-polar metabolites are dissolved in an organic solvent and polarmetabolites are dissolved in an aqueous solvent.
 22. The methodaccording to claim 21, wherein the extracted samples are analyzed byelectrospray ionization or atmospheric pressure chemical ionization. 23.The method according to claim 21, wherein the extracted samples areanalyzed by MS/MS transition.
 24. The method according to claim 21,wherein the extracted samples are analyzed by chromatography and MS/MStransition.
 25. The method according to claim 24, wherein the MS/MStransitions for the diacyl glycerylphosphatidylethanolamines are:718.0/255.0, 716.0/255.0, 746.0/283.0 and 744.0/283.0, respectively. 26.The method according to claim 24, wherein the MS/MS transitions for theplasmanyl glycerylphosphoethanolamines are: 702.0/281.0, 700.0/279.0,724.0/303.0, 752.0/331.0, 748.0/327.0, 730.0/281.0, 728.0/279.0,752.0/303.0, 780.0/331.0 and 776.0/327.0, respectively.
 27. The methodaccording to claim 24, wherein the MS/MS transitions for the plasmenylglycerylphosphoethanolamines are: 700.0/281.0, 698.0/279.0, 722.0/303.0,750.0/331.0, 746.0/327.0, 728.0/281.0, 726.0/279.0, 750.6/303.2,778.0/331.0 and 774.0/327.0, respectively.
 28. The method according toclaim 24, wherein the MS/MS transitions for the free fatty acids are:327.2/283.0 and 303.2/259.5, respectively.